The purpose of this proposal is to define the molecular mechanisms of an inhibitory Rac2 mutant, Rac/D57N. This mutation was identified in a patient with severe immunodeficiency. The specific of this proposal will examine the effects of this mutation on guanine nucleotide binding. Interactions with Rac regulators and effectors will be determined. Also, the effects of this mutation on Rac signaling in vivo will be examined 3H-GDP and 35S-GTPgammaS binding assays will be used to assess the intrinsic guanine nucleotide binding properties of Rac2/D57N relative to wild-type Rac2. Biochemical studies will use recombinant proteins expressed in bacteria and insect cells to study the in vitro interactions of Rac with smgGDS, RhoGDI, p67/phox, and PAK. These proteins represent upstream regulators of Rac/D57N will be examined in vivo by transfecting embryonic stem cells with either wild-type Rac2 of Rac/D57N. Positively transfected cells will be selected and differentiated into macrophages. The inhibitors of Rac2/D57N on superoxide production, regulation of the cytoskeleton, and Rac2 activated protein kinase cascades will be examined. This proposal will define the molecular mechanisms of an inhibitory Rac2 mutant recently defined in a human disease in our research laboratory.